The present disclosure relates to a mutant lactate oxidase having increased stability, a nucleic acid encoding the mutant lactate oxidase, an expression vector comprising the nucleic acid, a host cell comprising the nucleic acid or the expression vector, a method of determining lactate in a sample, the use of the mutant lactate oxidase for determining lactate, a device for determining lactate in a sample using the mutant lactate oxidase and a kit for determining lactate comprising the mutant lactate oxidase.
Lactic acid, also known as milk acid, plays a role in several biochemical processes. Lactic acid is an alpha hydroxy acid with the chemical formula C3H6O3. In solution it is present in its ionic form, i.e., as lactate CH3CH(OH)COO−. Lactate is chiral and has two optical isomers. One is known as L-(+)-lactate or (S)-lactic acid and the other is D-(−)-lactic acid or (R)-lactic acid. L-(+)-lactic acid is the biologically important isomer.
In animals (including humans), L-lactate is constantly produced from pyruvate via the enzyme lactate dehydrogenase (LDH) during normal metabolism and exercise. It normally does not increase in concentration until the rate of lactate production exceeds the rate of lactate removal, which is governed by a number of factors, including monocarboxylate transporters, concentration and isoform of LDH, and oxidative capacity of tissues. In humans, the concentration of blood lactate is usually 1-2 mmol/L at rest, but can rise to over 20 mmol/L during intense exertion.
The lactate concentration or lactate to pyruvate ratio reflects the redox state. Monitoring lactate levels is, therefore, a good indicator of the balance between tissue oxygen demand and utilization and is useful when studying cellular and animal physiology. Accordingly, lactate in biological samples such as serum, plasma, blood, urine, and saliva or intracellular and extracellular lactate concentrations in cell culture samples may be monitored in order to study or monitor a subject's or cell's condition.
Determinations of blood lactate levels are frequently done in the context of competitive sports, fitness, and rehabilitation, these determinations are used to:                assess the intensity of individual exercise routines;        improve various phases of exercise and recovery; and        reduce of the risk of overload and injury.        
As detailed above, anaerobic glycolysis markedly increases blood lactate, especially with prolonged exercise.
In medicine, the determination of lactate levels in a patent may be used to:                assay for tissue hypoxia; and        estimate the severity of disease and prognosis, in so far as it is reflected by abnormal levels of lactate measured in a patient.For example, lactate concentrations can be increased in any condition that decreases the amount of oxygen available to the body, increases lactate production, and/or decreases lactate clearance. This can be anything from localized increases of lactate in muscle due to strenuous exercise up to life-threatening systemic shock. Excess lactate may be present in a range of diseases, infections, and inherited metabolic and mitochondrial disorders. The common cause for increased blood lactate is anoxia resulting from conditions such as shock, pneumonia and congestive heart failure. Lactic acidosis may also occur in renal failure and leukemia. Also, thiamine deficiency and diabetic ketoacidosis are associated with increased levels of lactate. They may also be caused by certain medications, such as metformin (taken by diabetics) and isoniazid (tuberculosis treatment).        
Tests for the determination of lactate are known in the art. In recent years, enzymatic methods for the determination of lactate have been developed. Enzymatic methods involve the use of an enzyme in the determination method, are generally simple and provide greater specificity, accuracy, and reproducibility than non-enzymatic. The first enzymatic method described for the determination of lactate was based on the transfer of hydrogen from lactate to potassium ferricyanide by lactate dehydrogenase. This procedure was cumbersome and did not receive wide acceptance. Subsequent methods involved the UV measurement of the formation of NADH. In 1974, a lactate procedure was described that measures the amount of NADH formed by the oxidation of lactate catalyzed by lactate dehydrogenase. This method uses hydrazine as a trapping agent for pyruvate. Another method is also based on the catalytic action of lactate dehydrogenase but includes alanine transaminase in the reaction mixture to more rapidly remove the pyruvate formed from the conversion of lactate. Still another method uses an enzymatic reaction to convert lactate to pyruvate involving lactate oxidase. The hydrogen peroxide produced by this reaction may be then used in an enzymatic reaction to generate a colored dye.
The term “lactate oxidase” (classified as EC 1.1.3.15 by the Enzyme Commission of the International Union of Biochemistry) generally means an enzyme that catalyses the oxidation of L-lactate to pyruvate with reduction of O2 to H2O2 ((S)-2-hydroxy-acid oxidase). Lactate oxidase is a member of a family of FMN (flavin mononucleotide)-dependent alpha hydroxy acid oxidizing enzymes. It employs flavin mononucleotide (FMN) as cofactor. Lactate oxidase enzymes appear in viruses and cellular organisms.
Lactate oxidase from Aerococcus viridans is often used in biosensors and in vitro tests in order to detect lactate, e.g., in blood. These biosensors and in vitro tests are predominantly used in the monitoring of, e.g., intensive care patients and athletes. The lifetime of the sensors is determined by the stability of the lactate oxidase which has a finite shelf-life and becomes inactivated during use. The stability of lactate oxidase also influences the shelf-life of lactate oxidase when it is used as a reagent or as part of a device. The stability of lactate oxidase also influences the in-use time of the enzyme when it is used as a reagent for in vitro testing. A variant of lactate oxidase that exhibits greater stability will have a longer shelf-life than a less stable variant. And one can use a more stable variant of lactate oxidase to perform a larger number of assays than one can expect to perform with a less stable variant of the enzyme.
Due to lactate oxidase's limited shelf-life and relatively short half-life when it is used to measure lactate as part of a device such as a sensor or as a reagent in a test, it is frequently necessary to have to substitute the lactate oxidase with additional lactate oxidase in the device or in the assay, as the activity of the enzyme falls below a technically acceptable level. Frequent changes of sensors and test equipment is consumer-unfriendly, a waste of resources and, therefore, to be avoided. Due to the low stability of the wild-type lactate oxidase, determinations involving lactate oxidase are usually carried out at reduced temperature. Accordingly, an additional advantage of a variant of lactate oxidase with increased stability would be that within devices used for applications such example blood analysis it would not be as critical to have a thermostat for maintaining the temperature of the enzyme in, for example, the 25 to 30° C. range. This is especially useful because blood gas analysis (often carried out concomitantly) is usually carried out at 37° C. Accordingly, a more stable lactate oxidase would significantly reduce complexity of devices, thereby providing a basis for low-cost devices, which is of particular relevance, e.g., in so-called emerging markets. Therefore, it is of great interest to find ways to increase stability of the lactate oxidase in order to increase its shelf-life and its in-use time when used in biosensors and in vitro tests.